1 Introduction:

The purpose of this tutorial is to provide a step-by-step guide to isolate nuclei from flash-frozen tissue for single nucleus RNA sequencing. Nuclei Isolation is the first step in many novel techniques including snNRAseq, single-nucleus assay for transposase-accessible chromatin sequencing, epigenomic analysis. Although based off of the 10X Genetics Chromium Nuclei Isolation Protocol, this tutorial is specifically for the use of kidney tissue for snRNAseq and includes alterations to maximize yield and quality. This tutorial will also include a guide on how to assess and grade the quality of the isolated nuclei as well as next steps after the initial isolation.

2 Resources from 10X Genetics:

Link to 10X Protocol, starts on Page 25

Edited Visual Diagram of Protocol, with inserted notes for easy reading:

3 Protocol:

The protocol includes four main steps: buffer set up, lysis, debris removal, and resuspension.

3.1 Buffer Set-Up

Table of Buffers:

#install.packages("openxlsx")
#install.packages("here")
    library(openxlsx)
    library(here)
## here() starts at /Users/debs/Nelson Lab
library(openxlsx)
library(dplyr)
## 
## Attaching package: 'dplyr'
## The following objects are masked from 'package:stats':
## 
##     filter, lag
## The following objects are masked from 'package:base':
## 
##     intersect, setdiff, setequal, union
library(knitr)
library(kableExtra)
## 
## Attaching package: 'kableExtra'
## The following object is masked from 'package:dplyr':
## 
##     group_rows
library(here)

# Read the data
df <- read.xlsx(here("buffers.xlsx"))
url1 <- df$url1  


df2 <- df %>% select(-url1) %>% 
  mutate_all(~ replace(., is.na(.), ""))
## Warning: Using an external vector in selections was deprecated in tidyselect 1.1.0.
## ℹ Please use `all_of()` or `any_of()` instead.
##   # Was:
##   data %>% select(url1)
## 
##   # Now:
##   data %>% select(all_of(url1))
## 
## See <https://tidyselect.r-lib.org/reference/faq-external-vector.html>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
df3 <- df2 %>%
  kbl(booktabs = T, align = "c") %>%
  kable_styling() %>%
  column_spec(1, bold = T, color = "#990000") %>%  
  column_spec(1:ncol(df2), width = "12em") %>%    # Set all columns to same width
  row_spec(1:nrow(df2), align = "c" ) %>%    # Bold the top row (header) with red color
  row_spec(3:nrow(df2), align = "c")               # Align the rest of the rows to center


df3
Buffer.Name Solute/.Component.1 Solvent/.Component.2 Solvent/.Compound.3
1X PBS 10 mL of 10X PBXS 90 mL DI water N/A
10% BSA 10 g BSA powder 100 mL DI water N/A
Lysis Buffer Reducing Agent B (PN:2000558) Surfactant A (PN: 2000559) Lysis Reagent (PN: 2000558)
Debris Buffer Reducing Agent B (PN:2000558) N/A Debris Removal Reagent (PN: 2000560)
Wash and Resuspention Buffer 1X PBS 10% BSA Rnase Inhbitor (PN: 2000565)

Start with the Wash and Resuspension Buffer, which is composed of the 1X PBS and 10% BSA solutions that are not included in the kit. Generally, make a stock of 100 mL of each and keep chilled at 4 degrees Celcius. Refer to the table above and the instructions for components and amounts.

1X PBS: Dilute 10 mL into 90 mL of Ultra-Pure Water. Invert to mix thoroughly.
10% BSA: 10 grams of powder BSA to 100 mL of Ultra-Pure Water. Mix gently by swirling to prevent excess frothing.

Next, retrieve all components of the 10X Nuclei Isolation Kit, which should include the Lysis Reagent, Debris Removal Reagent, Reducing Agent B, Surfactant A, and RNAse Inhibitor. Also retrieve the Sample Dissociation Tube from the room temperature Consumables portion.

Place everything except for the Reducing Agent B on ice and begin to prepare the buffers. After Reducing Agent B thaws and everything is briefly vortexed, begin preparing the buffers.

Per reaction, the Lysis Buffer requires 550 µl Lysis reagent, 0.55 µl Reducing Agent B, and 5.5 µl Surfactant A. Per reaction, the Debris Removal Buffer requires 550 µl Debris Removal Reagent and 0.55 µl of the Reducing Agent B. Per reaction, the Wash and Resuspension Buffer requires 2,887.5 µl 1X PBS, 330 µl 10% BSA, and 82.5 µl of RNase Inhibitor for sequencing.

Keep all completed buffers labeled and on ice. Pre-chill the centrifuge to 4 degrees C.

3.2 Main Procedure

Next, retrieve flash frozen half kidney from the -80 degrees storage. While frozen, use tweezers or scissors to remove the kideny and place it in a pre-chilled Sample Dissociation Tube. Add 200 µl of the Lysis Buffer and wait for the kidney to mostly thaw. Using sterile scissors, chop up the kidney into small pieces, closing and opening scissors at least 50 times.

Then using the green pestle provided in the room-temperature pack, grind up the tissue, pressing down and twisting at least 20 times or until the solution is smooth with no visible chunks.

Next, add 300 µl of the Lysis buffer and pipette up and down, making sure that there are no chunks that block the pipette tip. Incubate the solution on ice for 8 minutes, starting the timer after completing the grinding step.

Next, pipette the dissociated tissue solution onto the Nuclei Isolation Column in the collection Tube (both included in room temperature Consummables) and spin in the centrifuge for 16,000 rcf, 20 sec, at 4 degrees C. Make sure that this cycle is evenly balanced in the centrifuge, because of the high speed.

After the cycle ends, discard the column and vortex the flow-through for 10 seconds to resuspend the nuclei. Then spin in the centrifuge at 700 rcf, 3 min, 4 degrees C. Make sure that when placing the Nuclei Isolation Column, position it within the centrifuge so that the hinge of the lid points out, the front tab facing in. This will make it easier to find the pellet and avoid losing nuclei.

Next, remove the supernatent, being careful to avoid the pellet and angling the pipette tip away from the hinge side of the column. Resuspend the pellet with 500 µl of the Debris Removal Buffer. Spin at 700 rcf for 10 minutes at 4 degrees C. While waiting, mix the final Resuspension Buffer, which should be 0.04% BSA in PBS.

Remove the supernatant again and resuspend in 1 mL of the Wash and Resuspension Buffer. Spin at 500 rcf for 5 minutes, 4 degrees C. Remove supernatant, resuspend again in 1 mL of Wash and Resuspension Buffer. Spin again at 500 rcf for 5 minutes, 4 degrees C.

Finally, resuspend the. nuclei pellet in 500 µl of the Final Wash and Resuspension Buffer (0.04% BSA). Vortex the solution for 3 seconds and let it sit undisturbed for 15 minutes.

Now, depending on QC or following uses, use the concentrated solution.

3.3 Nuclei Count with Light Microscope:

With the nuclei solution, add 2 µl to 18 µl of Trypan Blue, pipetting up and down into the TB at least 10 times to mix thoroughly. Note that while Trypan Blue does not aggregate in whole cells, the compound does aggregate within the exposed nuclei, which is what we will be checking for. Add 10 µl to one of the wells on a hemocytometer, making sure the solution fills the space. Observe on a light microscope, counting to get an approximation and assessment of nuclei quality.

Microscope used in this procedure:

link to hemocytometer counter app

From this procedure, the average expected nuclei concentration count is: 500 nuclei per microliter This is approximately 50 to 60 nuclei per quadrant in the hemocytometer.

3.4 Nuclei Quality Check:

Priority Check:

  1. No clumps, with an even distribution

  2. Quality:

A grade Nuclei: Good, circular shape with no blebbing, halo of Trypan Blue around the edges that suggest an undisturbed nuclear envelope. No aggregation.

B grade Nuclei: Slightly irregular shapes, still has the halo but less bright or more uneven

C grade Nuclei: Losing more of normal shape, small blebbing, paler color

D/F grade Nuclei: Complete loss of structure, no blue halo, aggregates

Example of a graded sample:

Example of grading with reasoning. Note: this is an image of an aggregate that was included because of a wide variety of quality, but as a general rule aggregates indicate a low quality overall sample.

Example of a sample viewed at 10x and 40x, grading the individual nuclei quality is easier at 40x but it is recommended to conduct overall sample quality checks at 10x magnification.